Pyridine classical cannabinoid compounds and related methods of use

ABSTRACT

Disclosed are compounds of the formula I: 
                         
wherein R 1 , R 2 , V, W, X, Y and Z can be as defined herein. The compounds can be used in the treatment of disorders mediated by the cannabinoid receptors.

This application is a continuation-in-part of and claims prioritybenefit from application Ser. No. 12/468,773 filed May 19, 2009, nowU.S. Pat. No. 8,124,771 issued Feb. 28, 2012 which claims priority fromapplication Ser. No. 61/128,160 filed May 19, 2008, each of which isalso incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

The classical cannabinoid, delta-9-tetrahydrocannabinol (Δ⁹-THC), is themajor active constituent extracted from Cannabis sativa. The effects ofcannabinoids are due to an interaction with specific high-affinityreceptors. Presently, two cannabinoid receptors have been characterized:CB-1, a central receptor found in the mammalian brain and a number ofother sites in the peripheral tissues; and CB-2, a peripheral receptorfound principally in cells related to the immune system. In addition, ithas recently been reported that the GPR35 and GPR55 orphan receptorsbind cannabinoid type ligands and have been proposed as third receptorsubtypes. The CB-1 receptor is believed to mediate the psychoactiveproperties associated with classical cannabinoids. Characterization ofthese receptors has been made possible by the development of specificsynthetic ligands such as the agonists WIN 55212-2 (D'Ambra et al., J.Med. Chem. 35:124 (1992)) and CP 55,940 (Melvin et al., Med. Chem. 27:67(1984)).

Pharmacologically, cannabinoids can be used to affect a variety oftargets such as the central nervous system, the cardiovascular system,the immune system and/or endocrine system. More particularly, compoundspossessing an affinity for either the CB-1 or the CB-2 receptors andpotentially the GPR35 and GPR55 receptors are useful as anticanceragents, antiobesity agents, analgesics, myorelaxation agents andantiglaucoma agents. Such compounds can also be used for the treatmentof thymic disorders, vomiting; various types of neuropathy, memorydisorders, dyskinesia, migraine, multiple sclerosis; asthma, epilepsy,ischemia, angor, orthostatic hypotension, osteoporosis, liver fibrosis,inflammation and irritable bowel disease, and cardiac insufficiency.

However, certain cannabinoids such as Δ⁹-THC also affect cellularmembranes, producing undesirable side effects such as drowsiness,impairment of monoamine oxidase function, and impairment of non-receptormediated brain function. The addictive and psychotropic properties ofsome cannabinoids tend to limit their therapeutic value.

There still remains an ongoing need in the art for compounds, whetherclassical or non-classical cannabinoid analogs, that can be used fortherapeutic purposes to affect treatment of conditions or disorders thatare mediated by the CB-1 receptor and/or the CB-2 receptor.

SUMMARY OF THE INVENTION

In light of the foregoing, it is an object of the present invention toprovide a range of heterocyclic cannabinoid analog compounds,compositions and/or related methods, thereby overcoming variousdeficiencies and shortcomings of the prior art, including those outlinedabove. It will be understood by those skilled in the art that one ormore aspects of this invention can meet certain objectives, while one ormore other aspects can meet certain other objectives. Each objective maynot apply equally, in all its respects, to every aspect of thisinvention. As such, the following objects can be viewed in thealternative with respect to any one aspect of this invention.

It can be an object of the present invention to identify one or moreclasses of cannabinoid compounds exhibiting affinity for cannabinoid andrelated receptors found in human cells and tissues.

It can also be an object of the present invention to provide one or morenovel pyridine classical cannabinoid receptor ligands, such compoundscomprising a pyridine ring system substitution in the A-ring, suchcompounds as can comprise all known and inferred C3 side chainsubstitutions together with a hexahydro-, tetrahydro- or non-pyrane ringsystem.

It can be another object of the present invention to identify suchcompounds exhibiting cannabinoid receptor selectivity, for directedtherapeutic use.

Other objects, features, benefits and advantages of the presentinvention will be apparent from this summary and the followingdescriptions of certain embodiments, and will be readily apparent tothose skilled in the art having knowledge of various cannabinoidcompound and related therapeutic methods. Such objects, features,benefits and advantages will be apparent from the above as taken intoconjunction with the accompanying examples, data, figures and allreasonable inferences to be drawn therefrom, alone or with considerationof the references incorporated herein.

In part, the present invention can be directed to a cannabinoid analogcompound selected from compounds of a formula I below

wherein one of W and X can be N and the other can be C; ----- representsan optional double bond wherein the ring that optionally contains it canbe selected from hexahydro, 6a,10a-dehydro, 8,9-dehydro, and9,10-dehydro; Y can be selected from S, O, CH₂, CH(CH₃), CH(OH),C(CH₃)(OH), C(CH₃)₂, C(—V(CH₂)_(n)V—), C(O), NH, S(O), and S(O)₂; V canbe selected from CH₂, S and O; n can be an integer ≧1, and preferablyfrom 1 to 6; Z can be selected from H, substituted and unsubstitutedalkyl, substituted and unsubstituted cycloalkyl, substituted andunsubstituted aryl, substituted and unsubstituted heteroaryl,substituted and unsubstituted heterocycloalkyl, and arylalkyl,cycloalkylalkyl, heteralkylalkyl and heteroarylalkyl, wherein each alkylportion can be optionally substituted up to three times and the ringportion of each can be optionally substituted with one, two, three, fouror five substituents; and R₁ can be selected from H and unsubstituted orsubstituted alkyl, including but not limited to aminoalkyl,morpholinoalkyl, and hemisuccinate moieties.

In part, the present invention can be directed to a salt of a compoundin accordance herewith.

In part, the present invention can be directed to a pro-drug of acompound in accordance herewith.

In part, the present invention can also be directed to a pharmaceuticalcomposition comprising a compound of the sort described herein, a saltand/or a pro-drug thereof, and a pharmaceutically acceptable carriercomponent.

In part, the present invention can be directed to a method of modifyingthe activity of a cannabinoid receptor. Such a method can compriseproviding a compound, salt and/or pro-drug of the present invention orany other compound disclosed herein that has activity at a cannabinoidor related receptor, a salt and/or pro-drug thereof; and contacting acell and/or cannabinoid receptor of a cell with such a compound. Asillustrated below, such contact can be at least partially sufficient toat least partially modify activity of such a cannabinoid receptor.

In part, the present invention can also be directed to a method oftreating a cannabinoid receptor-mediated condition. Such a method cancomprise providing a compound in accordance herewith or any othercompound disclosed herein that has activity at a cannabinoid receptor, asalt and/or pro-drug thereof; and administering to a patient an amountof such a compound, salt and/or pro-drug, that is at least partiallyeffective to treat a cannabinoid receptor-mediated condition. Thisaspect of the invention can relate to the use of agonists of a CB-1 or arelated receptor, antagonists of a CB-1 or related receptor, agonists ofa CB-2 or related receptor, and/or antagonists of a CB-2 or relatedreceptor to treat or prevent disease conditions mediated byhyperactivity of CB-1 and/or CB-2 (or related) receptors or eitherinactivity or hypoactivity of the CB-1 and/or CB-2 (or related)receptors.

In part, the present invention can also be directed to a compoundselected from compounds of a formula

wherein the C-ring can comprise or be selected from hexahydro-,6a,10a-dehydro-, 8,9-dehydro- and 9,10-dehydro-structures; R₁ can beselected from H, alkyl, aminoalkyl, morpholinoalkyl, and hemisuccinatemoieties; Y can be selected from carbonyl, dimethylmethylene andhydroxymethylene moieties; and Z can be selected from substituted andunsubstituted alkyl, phenyl, substituted phenyl, cycloalkyl, substitutedcycloalkyl, thiophenyl and substituted thiophenyl moieties, suchsubstituents as would be understood by those skilled in the art madeaware of this invention, including but not limited to those describedelsewhere herein. In certain embodiments, Z can be an alkyl, phenyl orcycloalkyl moiety and, optionally, Y can be a dimethylmethylene moiety.Regardless, such a compound can be selected from salts and/or pro-drugsof such a compound.

Without limitation, this invention can also be directed to a method ofcancer treatment. Such a method can comprise providing a cancer cellcomprising a cannabinoid receptor, such a cell of a growth of cancercells; and contacting such a growth with a cannabinoid compound selectedfrom compounds of a formula I

wherein the C-ring, R₁, Y and Z can be as described above. In anembodiment, R₁ can be selected from H, alkyl, alkylamino,alkylmorpholino and hemisuccinate moieties; Y can be selected fromcarbonyl, dimethylmethylene and hydroxymethylene moieties; and Z can beselected from substituted and unsubstituted alkyl, phenyl, substitutedphenyl, cycloalkyl, substituted cycloalkyl, thiophenyl and substitutedthiophenyl moieties, with such substituents as would be understood bythose skilled in the art made aware of this invention, including but notlimited to those described elsewhere herein, and salts and pro-drugs ofsaid compounds, and combinations thereof, such compound(s) in an amountat least partially sufficient to induce death of a cell of such agrowth. In certain embodiments, Z can be selected from substitutedalkyl, unsubstituted alkyl, substituted cycloalkyl, unsubstitutedcycloalkyl, phenyl and substituted phenyl moieties, with suchsubstituents as can be selected from chloro, hydroxy and methoxymoieties. In certain such embodiments, R₁ can be selected from H andmethyl moieties. Regardless, without limitation and as illustratedelsewhere herein, Y can be dimethylmethylene or carbonyl.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the functional activity of compound 6b at the CB-1receptor.

FIG. 2 shows the secretion profiles of G-CSF by A549 cells exposed tocompound 6b at the EC1 and EC10 in the presence and absence of TNF-α at4 and 18 hour intervals.

FIG. 3 shows the secretion profiles of IL-1β by A549 cells exposed tocompound 6b at the EC1 and EC10 in the presence and absence of TNF-α at4 and 18 hour intervals.

FIG. 4 shows the secretion profiles of IL-6 by A549 cells exposed tocompound 6b at the EC1 and EC10 in the presence and absence of TNF-αscaled to show the levels at the 18 hour interval.

FIG. 5 shows the secretion profiles of IL-6 by A549 cells exposed tocompound 6b at the EC1 and EC10 in the presence and absence of TNF-αscaled to show the levels at the 4 hour interval.

FIG. 6 shows the secretion profiles of IL-8 by A549 cells exposed tocompound 6b at the EC1 and EC10 in the presence and absence of TNF-αscaled to show the levels at the 18 hour interval.

FIG. 7 shows the secretion profiles of IL-8 by A549 cells exposed tocompound 6b at the EC1 and EC10 in the presence and absence of TNF-αscaled to show the levels at the 4 hour interval.

FIG. 8 shows the secretion profiles of MCP-1 by A549 cells exposed tocompound 6b at the EC1 and EC10 in the presence and absence of TNF-α at4 and 18 hour intervals.

FIG. 9 shows the secretion profiles of MIF by A549 cells exposed tocompound 6b at the EC1 and EC10 in the presence and absence of TNF-αscaled to show the levels at the 4 hour interval.

FIG. 10 shows the secretion profiles of MIF by A549 cells exposed tocompound 6b at the EC1 and EC10 in the presence and absence of TNF-αscaled to show the levels at the 18 hour interval.

FIG. 11 shows the secretion profiles of RANTES by A549 cells exposed tocompound 6b at the EC1 and EC10 in the presence and absence of TNF-αscaled to show the levels at the 18 hour interval.

FIG. 12 shows the secretion profiles of RANTES by A549 cells exposed tocompound 6b at the EC1 and EC10 in the presence and absence of TNF-αscaled to show the levels at the 4 hour interval.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

The novel compounds encompassed by the instant invention are thosedescribed by the general Formula I set forth above, and thepharmaceutically acceptable salts and prodrugs thereof.

By “alkyl” in the present invention is meant straight or branched chainalkyl radicals having from 1-20 carbon atoms. Optionally, an alkyl groupof the instant invention can contain one or more double bonds and/or oneor more triple bonds.

By “cycloalkyl” is meant a carbocyclic radical having from three totwelve carbon atoms. The cycloalkyl can be monocyclic or a polycyclicfused system. Optionally, a cycloalkyl group of the instant inventioncan contain one or more double bonds and/or one or more triple bonds.

The term “heterocyclyl” refers to one or more carbocyclic ring systemsof 4-, 5-, 6- or 7-membered rings which includes fused ring systems andcontains at least one and up to four heteratoms selected from nitrogen,oxygen or sulfur and combinations thereof.

By “aryl” is meant an aromatic carbocyclic ring system having a singlering, multiple rings or multiple condensed rings in which at least onering is aromatic.

The term “heteroaryl” refers to one or more aromatic ring systems havingfrom three to twelve atoms which includes fused ring systems andcontains at least one and up to four heteroatoms selected from nitrogen,oxygen or sulfur and combinations thereof.

By “arylalkyl” is meant an alkyl radical substituted with an aryl, withthe the point of attachment is a carbon of the alkyl chain.

As used herein, “substituted” refers to those substituents as would beunderstood by those skilled in the art. At least one and as many as fivesubstituents can exist on a single group. Examples of such substituentsinclude, but are not limited to, halo, alkyl, alkoxy, hydroxyl, aryl,heteroaryl, cycloalkyl, heterocycloalkyl, cyano, nitro, amino,alkylamino, dialkylamino, thiol, alkylthiol, haloalkyl (e.g.trifluoromethyl), carboxy, alkylcarboxy, carbamoyl and the like.

According to one approach, representative, non-limiting pyridine analogscan be prepared by reacting an intermediate pyridine with theappropriate terpine to generate the described C-rings according to theSchemes shown below. The hexahydro is synthesized utilizing amodification of the tandem cyclization method described by Tietz, Chem.Rev., 96:115-136 (1996), and the references cited therein, allincorporated herein by reference in their entirety. The ring formationsare accomplished under microwave conditions with the appropriatelysubstituted pyridine and citronellal, either racemic or opticallyactive, as depicted in Scheme 1.

The synthesis of the 9,10- and 8,9-dehydro analogs is accomplished underthe same reaction conditions as the hexahydro, however, citronellal issubstituted with TBS protected 3-hydroxyl citronellal (Scheme 2). Thesynthesis of this compound has previously been reported by Kesenheimerand Groth, Org. Lett., 8:2507 (2006), incorporated herein by referencein its entirety.

Synthesis of the 6a,10a-dehydro is accomplished using a method of Adams(U.S. Pat. No. 2,419,934 as incorporated herein by reference in itsentirety) utilizing racemic or optically active pulegone and theappropriately substituted pyridine analog (Scheme 3).

The corresponding pyridines are prepared by reacting dimethyl-,diethyl-, or bis(trichlorophenyl)-malonates with the appropriatelysubstituted Schiff's base derived from the requisite 2-keto analogs,Scheme 4 (See, Ito and Miyajima J., Heterocycli Chem., 29:1037 (1992),and Kappe et al., J. Heterocyclic Chem., 25:463 (1988), each of which isincorporated herein by reference in its entirety), wherein R₂ is benzylor t-butyl and R₃ and R₄ are methyl, ethyl, phenyl, and/orbis(trichlorophenyl).

The requisite substituted 2-keto compounds are either selected fromcommercially available materials or synthesized from the appropriatelysubstituted nitrile using methyl magnesium bromide or methyl lithium(Scheme 5). Alternatively, the imine formed by the reaction with methyllithium can be used directly in the formation of the pyridine ringsystem.

The starting nitriles are derived from commercially available materialsor synthesized using the methods shown in Schemes 6 and 7 which arerepresentative of but not limited to the scope of the chemistry.Derivatives containing a gem-dialkyl, heterocyclic, or carbocyclicsubstituent at Y, where commercial compounds are not available, areprepared either by direct alkylation of the methylene nitrile (See, U.S.Pat. No. 7,057,076 to Makriyannis and Pub. No. 2004/087590, each ofwhich is incorporated herein by reference in its entirety) or from theappropriately substituted aryl, heteroaryl halogen and isopropylnitrile.

Derivatives containing a keto, hydroxyl, alkylhydroxyl substituent at Ycan be prepared by direct oxidation of compounds bearing a Y═CH₂ or fromthe C2-aldehyde pyridine, prepared from bis-ethylsulfanyl-acetaldehyde(Scheme 8) using chemistry previously reported in U.S. Pat. No.7,169,942, the entirety of which is incorporated herein by reference.

While syntheses of several representative, non-limiting compounds aredescribed herein, it will be understood by those skilled in the art thatvarious other compounds can be prepared using similar such proceduresand/or straight-forward modifications thereof. Accordingly, theidentities of moieties R₁, Y and Z are limited only by the respectivereagents, starting materials, intermediates and chemistry thereon.

Likewise, the present invention contemplates, more broadly, variousother such compounds, salts and/or pro-drugs thereof, together withcorresponding pharmaceutical compositions thereof, as also described inthe aforementioned co-pending application. Such compounds, salts,pro-drugs and/or pharmaceutical compositions can be used as describedtherein. For instance, the present invention can be used to modify theactivity of one or both of the CB-1 and CB-2 receptors. Such a methodcan be carried out by contacting a cell and/or cannabinoid receptorthereof with a compound of the present invention, such contact at leastpartially sufficient to at least partially modify the activity of such acannabinoid receptor, whether ex vivo or in vivo.

More generally, various physiological and/or therapeutic advantages ofthe present compounds and/or compositions can be realized withconsideration of the authorities cited in the aforementioned co-pendingapplication. The inventive analogs, as described herein, can beadministered in therapeutically-effective amounts to treat a wide rangeof indications. Without limitation, various such conditions and/ordisease states are described in paragraph 0067 of co-pending applicationSer. No. 12/074,342, filed Mar. 3, 2008 and entitled“Tri-Aryl/Heteroaromatic Cannabinoids and Use Thereof,” the entirety ofwhich is incorporated herein by reference.

Accordingly, this invention can be directed to a method comprisingproviding a compound of the sort described herein, such a compoundexhibiting activity at a cannabinoid receptor; and contacting a cellcomprising a cannabinoid receptor with such a compound and/oradministering such a compound to a patient, such a compound in an amountat least partially effective to treat a cannabinoid receptor/mediatedcondition. Such a cannabinoid receptor can be a receptor describedherein or as would otherwise be understood or realized by those skilledin the art made aware of this invention.

The activity of cannabinoid and related receptors can be affected,mediated and/or modified by contacting such a receptor with an effectiveamount of one or more of the present compounds as can be present in oras part of a pharmaceutical composition or treatment, or by contacting acell comprising such a receptor with an effective amount of one or moresuch compounds, so as to contact such a receptor in the cell therewith.Contacting may be in vitro or in vivo. Accordingly, as would beunderstood by those skilled in the art, “contact” means that acannabinoid and/or related receptor and one or more compounds arebrought together for such a compound to bind to or otherwise affect ormodify receptor activity. Amounts of one or more such compoundseffective to modify and/or affect receptor activity can be determinedempirically and making such a determination is within the skill in theart.

Without limitation, analog compounds of this invention can be used exvivo in receptor binding assays of the sort described in Example 2 ofthe aforementioned co-pending '342 application. In vitro activity of thepresent analog compounds can be demonstrated in a manner similar to thatdescribed in Example 3 of the co-pending application. Alternatively, invivo activity can be demonstrated using the protocols described inExamples 4 and 6, thereof. More specifically, anti-cancer activity ofvarious representative compounds of this invention can be shown againsthuman lung, prostate, colorectal and pancreatic cancer cell lines usingthe methodologies described in Example 9 of the aforementionedco-pending '342 application. Extending such a methodology, the presentinvention can also be used to treat cancer growth of the central nervoussystem and/or induce cellular death within such growth. In accordancewith this invention, various cannabinoid compounds of the sort describedherein, including but not limited to those discussed above, can also beused in conjunction with a method to treat human glioma and/or braincancers. Illustrating such embodiments, one or more compounds of thepresent invention can be provided and used, as described in theco-pending application, to contact and/or treat human brain cancers,such contact and/or treatment as can be confirmed by cell death and/orrelated effects.

EXAMPLES OF THE INVENTION

The following non-limiting examples and data illustrate various aspectsand features relating to the compounds, compositions and/or methods ofthe present invention, including the synthesis of pyridine classicalcannabinoid receptor ligands and/or compounds, as are available thoughthe methodologies described herein. In comparison with the prior art,the present compounds and methods provide results and data which aresurprising, unexpected and contrary thereto. While the utility of thisinvention is illustrated through the preparation and use of severalcompounds, moieties and/or substituents thereof, it will be understoodby those skilled in the art that comparable results are obtainable withvarious other compounds, moieties and/or substituents, as arecommensurate with the scope of this invention. All compounds are namedusing ChemBioDraw Ultra Version 11.0.01.

Example 1a

2-Methyl-2-(thiophen-2-yl)propanenitrile—To a solution of2-(thiophen-2-yl) acetonitrile (1 g, 8.13 mmol) in 4 ml anhydrous THF,KHMDS (24.4 mmol, 48.9 ml, 0.5M in toluene) was added at 0° C. Themixture was allowed to stir for 3 minutes, after which a solution of16.26 mmol iodomethane (1.13 ml in 26 ml anhydrous THF) was added slowlyover a period of 10 minutes. The mixture was stirred for 5 minutes andmonitored by TLC. Upon completion, the reaction was quenched withaqueous ammonium chloride. The organic phase was separated with ethylacetate and dried over sodium sulfate. The product was purified viavacuum distillation. (bp 42° C. at 1 torr) Yield: 89%. ¹H NMR (500 MHz,CDCl₃): δ (ppm) 7.4 ppm (d, 1H), 7.2 ppm (t, 1H), 7.0 ppm (d, 1H), 1.9ppm (s, 6H).

Example 1b

In a similar fashion the following compound was synthesized.

2,2-Dimethyloctanenitrile—Purified via vacuum distillation (bp 50-55° C.at 1.1 torr). Yield: 84% I.R. (neat) nitrile 2230 cm⁻¹, ¹H NMR (500 MHz,CDCl₃): δ (ppm) 1.5 ppm (m, 4H). 1.4-1.3 ppm (m, 12H), 0.9 ppm (s, 3H).

Example 2a

2-Methyl-2-phenylpropanenitrile—To a solution of fluorobenzene (5.85 mL,62 4 mmol) in 100 mL of anhydrous toluene was added isobutyronitrile(22.5 mL, 250 mmol) followed by 200 mL (100 mmol) of a 0.5 M solution ofKHMDS in toluene. The reaction was stirred at 80° C. for 24 hours. Thereaction was then allowed to cool to room temperature, diluted withdiethyl ether, and washed with water and brine. The organic fraction wasdried over sodium sulfate and concentrated under reduced pressure. Theproduct was purified by flash chromatography using an ethylacetate/hexanes gradient to yield 4.57 g (50%) of the objective compoundas a brown oil. MS: (ESI, Pos) m/z 168.0 (M+23) ¹H NMR (500 MHz, CDCl₃):δ (ppm) 7.48 (d, 2H), 7.39 (t, 2H), 7.31 (t, 1H), 1.73 (s, 6H).

Example 2b

In a similar fashion the following compound was synthesized.

2-Methyl-2-pyridin-2-yl-propanenitrile—Purified in a manner similar to2-methyl-2-phenylpropanenitrile 2a using 2-bromopyridine as the startingmaterial to yield a brown oil. MS: (ESI, Pos) m/z 168.9 (M+23).

Example 3a

3-Methyl-3-phenylbutan-2-one—To a solution of2-methyl-2-phenyl-propanenitrile (2a, 500 mg, 3 1 mmol) in anhydrous THFcooled to 0° C. was added methyl magnesium bromide (408 mg, 3.4 mmol).The reaction was warmed to room temperature and then refluxed overnight.The mixture was treated with 1N HCl and the aqueous phase extracted withdiethyl ether. Product was confirmed by MS: (ESI, Pos) m/z 187.2 (M+23).

Example 3b

Various other ketones can be prepared from the respective nitriles,using synthetic procedures comparable to those described above, toprovide the corresponding Schiffs base components en route to the Y-and/or Z-substituted pyridine intermediates, as illustrated below.

Example 4

6-Methylpyridine-2,4-diol—A solution of acetone (1 g, 17 2 mmol) andbenzylamine (1.7 g, 16 3 mmol) in benzene (40 mL) was added 4 g ofpowdered molecular sieves and the reaction was stirred at roomtemperature overnight. The sieves were filtered, the solvent removedunder reduced pressure, and the resulting residue was purified by columnchromatography. The imine is dissolved in diglyme to which 1.2equivalents of dialkyl or diaryl malonate was added and the mixturerefluxed for 24 hours. The product was separated by columnchromatography.

Example 5

6-Pentylpyridine-2,4-diol—Into a round bottomed flask fitted with aDean-Stark trap and condensor was added a solution of 2-heptanone (1 g,8 7 mmol) and benzylamine (0.9 g, 8.3 mmol) in benzene (40 ml). Themixture was refluxed overnight yielding approxiamtely 0.1 mL of water(64% yield of the imine based on colllected water). The solvent wasremoved and the imine purified by column chromatograph. The imine isdissolved in diglyme to which 1.2 equivalents of dialkyl or diarylmalonate was added and the mixture refluxed for 24 hours. The productwas separated by column chromatography.

Example 6a

(9R)-6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-isochromeno[4,3-c]pyridin-1-ol(6a)—Pyridine-2,4-diol (4, 80 mg, 0.72 mmol) was added to 4 mL ofabsolute ethanol in a 10 mL microwave reaction vessel. To this was added89 μL of pyridine and 4 μL of piperidine followed by 390 μL (2.16 mmol)of (R)-(+)-citronellal. The reaction vessel was then sealed andirradiated at 200 watts to 130° C. for 1 hour. The solvent was thenremoved by rotary evaporation and the product purified by flashchromatography using a methanol/methylene chloride gradient to yield 76mg (63%) of the objective product 6a as a light yellow solid. MS: (ESI,Pos) m/z 248.0 (M+1) ¹H NMR (500 MHz, CDCl₃): δ (ppm) 11.99 (br.s, 1H),7.11 (d, 1H), 5.84 (d, 1H), 3.33 (d, 1H), 2.34 (m, 1H), 1.83 (m, 2H),1.63 (m, 2H), 1.38 (s, 3H), 1.26 (m, 1H), 1.09 (s, 3H), 1.03 (m, 1H),0.94 (d, 3H), 0.58 (q, 1H) ¹³C NMR (500 MHz, CDCl₃): δ (ppm) 165.73,162.95, 132.42, 110.29, 101.93, 79.53, 48.64, 37.25, 35.68, 34.71,32.58, 27.96, 27.57, 22.68, 19.49.

Example 6b

In a similar fashion the following compounds were synthesized.

(6aS,9R,10aR)-6,6,9-trimethyl-3-(2-methyloctan-2-yl)-6a,7,8,9,10,10a-hexahydro-6H-isochromeno[4,3-c]pyridin-1-ol(6b) Off white waxy solid, Yield: 38% MS: (ESI, Pos.) 372.10 (M−1) ¹HNMR(300 MHz, DMSO-d6): ∂(ppm) 8.6 (s, 1H), 6.5 (s, 1H), 2.91 (m, 1H), 2.49(m, 1H), 2.31 (m, 2H), 1.28-1.33 (m, 10H), 1.04-1.23 (m, 14H), 0.99-1.02(m, 3H), 0.89 (d, 3H), 0.83 (t, 3H).

Example 6c

(6aR, 9S,10aS)-6,6,9-trimethyl-3-(2-methyloctan-2-yl)-6a,7,8,9,10,10a-hexahydro-6H-isochromeno[4,3-c]pyridin-1-ol(6c) This compound was synthesized by substituting (R)-(+)-citronellalwith (S)-(−)-citronellal. Off white waxy solid, Yield: 36% MS: (ESI,Pos.) 372.20 (M−1) ¹HNMR (300 MHz, CDCl₃): ∂(ppm) 6.2 (s, 1H), 3.1 (m,1H), 2.43 (m, 1H), 1.28-2.10 (m, 12H), 1.04-1.23 (m, 14H), 0.99-1.02 (m,3H), 0.91 (d, 3H), 0.83 (t, 3H).

Example 6d

(6aS, 9R,10aR)-3-(2-cyclohexylpropan-2-yl)-6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-isochromeno[4,3-c]pyridin-1-ol(6d)—Off white waxy solid, Yield:33% MS: (ESI, Pos.) 370.20 (M−1) ¹HNMR(500 MHz, CDCl₃): ∂(ppm) 7.37 (s, 1H), 6.0 (s, 1H), 3.41 (d, 1H), 2.38(t, 1H), 1.53-1.85 (m, 13H), 1.43-1.15 (m, 9H), 1.08-1.02 (m, 8H), 0.92(d, 3H).

Example 6e

(6aR, 9S,10aS)-3-(2-cyclohexylpropan-2-yl)-6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-isochromeno[4,3-c]pyridin-1-ol(6e)—Off white waxy solid, Yield:31% MS: : (ESI, Neg) 370.1 (M−1) ¹HNMR(300 MHz, CDCl₃): ∂(ppm) 7.48(s,1H), 6.04(s,1H), 3.43(d,1H), 2.41(t,1H), 1.52-1.88(m,13H), 1.43-1.15(m,8H), 1.08-1.02(m,8H), 0.94 (d,3H),0.65(q,1H).

Example 6f

(6aS, 9R,10aR)-6,6,9-trimethyl-3-(2-phenylpropan-2-yl)-6a,7,8,9,10,10a-hexahydro-6H-isochromeno[4,3-c]pyridin-1-ol(6f)—white powder, Yield:52% MS: (ESI, Neg) 364.1 (M−1) ¹HNMR (300 MHz,CDCl₃): ∂(ppm) 7.79 (bs,1H), 7.28(m,5H), 5.89(s,1H), 3.21(m,1H), 2.31(m,1H), 1.85(m,1H),1.60-1.62(m,9H), 1.4 (s,6H), 1.18(m,2H), 0.95(d,3H),0.61(q,1H).

Example 6g

(6aR, 9S,10aS)-6,6,9-trimethyl-3-(2-phenylpropan-2-yl)-6a,7,8,9,10,10a-hexahydro-6H-isochromeno[4,3-c]pyridin-1-ol(6g)—white powder, Yield:44% MS: (ESI, Neg) 364.0 (M−1) ¹HNMR (300 MHz,CDCl₃): ∂(ppm) 7.41(m,5H), 7.1 (bs,1H), 6.12(s,1H), 3.39(m,1H), 2.41(m,1H), 1.89(m,1H),1.62-1.63(m,9H), 1.43 (s,6H), 1.20(m,2H), 0.99(d,3H),0.65(q,1H).

Example 7

6,6,9-Trimethyl-3-pentyl-tetrahydro-6H-isochromeno[3,4-b]pyridin-1-ol—Thematerial was synthesized from 6-pentylpyridine-2,4-diol 5 and TBSprotected 3-hydroxyl citronella (Kesenheimer and Groth, Org. Lett.,8:2507 (2006)) as described above. The TBS protecting group was removedusing TBAF and the compound was dehydrated employing a general acid toyield a mixture of separable isomers.

Example 8

6,6,9-Trimethyl-7,8,9,10-tetrahydro-6H-isochromeno[3,4-b]pyridin-1-ol(8)—Pyridine-2,4-diol (4, 100 mg, 0 9 mmol) was added to 3 mL ofabsolute ethanol in a 10 mL microwave reaction vessel. To this was alsoadded 66 μL of pyridine and 3 μL of piperidine followed by 0.46 g (3mmol) of (R)-(+)-pulegone. The reaction vessel was then sealed andirradiated at 300 watts to 130° C. for 1 hour. The solvent was thenremoved by rotary evaporation and the product purified by flashchromatography using a methanol/methylene chloride gradient to yield alight yellow solid. MS: (ESI, Neg) m/z 244.9 (M−1).

Example 9

While several compounds with tetrahydro and dehydro C-ring structuresare shown, other such compounds can be prepared to provide a range of Y-and/or Z moieties, such compounds limited only by the commercial orsynthetic availability of the corresponding pyridine and terpineintermediates. Likewise, R₁ can be varied depending on choice ofstarting material or subsequent chemistry on the resulting cannabinoidcompound.

Example 10

Receptor Binding Assays

Cell membranes from HEK293 cells transfected with the human CB-1receptor and membranes from CHO-K1 cells transfected with the human CB-2receptor were prepared. [³H]CP 55,940 having a specific activity of 120Ci/mmol was obtained from Perkin-Elmer Life Sciences, Inc. All otherchemicals and reagents were obtained from Sigma-Aldrich. The assays werecarried out in 96 well plates obtained from Millipore, Inc. fitted withglass fiber filters (hydrophilic, GFC filters) having a pore size of1.2μ. The filters were soaked with 0.05% polyethyleneimine solution andwashed 5× with deionized water prior to carrying out the assays. Thefiltrations were carried out on a 96 well vacuum manifold (MilliporeInc.), the filters punched out with a pipette tip directly intoscintillation vials at the end of the experiment, and the vials filledwith 5 ml scintillation cocktail Ecolite (+) (Fisher Scientific).Counting was carried out on a Beckmann Scintillation Counter modelLS6500. Drug solutions were prepared in DMSO and the radioligand wasdissolved in ethanol.

Incubation buffer: 50 mM TRIS-HCl, 5 mM MgCl₂, 2.5 mM EDTA, 0.5 mg/mlfatty acid free bovine serum albumin, pH 7.4.

Binding protocol for the CB-1 receptor: 8 μg of membranes (20 μl of a1:8 dilution in incubation buffer) was incubated with 5 μl of drugsolution (10⁻⁴M to 10⁻¹²M) and 5 μl of 5.4 nM [³H]CP 55,940 in a totalvolume of 200 μl for 90 mins at 30° C. Non-specific binding wasdetermined using 10 μM WIN55,212-2 (K_(i)=4.4 nM). The membranes werefiltered and the filters washed 7× with 0.2 ml ice-cold incubationbuffer and allowed to air dry under vacuum.

Binding protocol for the CB-2 receptor: 15.3 μg of membranes (20 μl of a1:20 dilution in incubation buffer) was incubated with 5 μl of drugsolution (10⁻⁴M to 10⁻¹²M) and 5 μl of 10 nM [³H]CP 55,940 in a totalvolume of 200 μl for 90 minutes at 30° C. Non-specific binding wasdetermined using 10 μM WIN55,212-2 (K_(i)=4.4 nM). The membranes werefiltered and the filters washed 7× with 0.2 ml ice-cold incubationbuffer and allowed to air dry under vacuum.

Data accumulation and statistical analysis: Varying concentrations ofdrug ranging from 10⁻⁴M to 10⁻¹²M were added in triplicate for eachexperiment and the individual molar IC₅₀ values were determined usingGraphPad Prism. The corresponding K_(i) values for each drug weredetermined utilizing the Cheng and Prusoff equation and final data waspresented as K_(i)±S.E.M. of n≧2 experiments.

Functional assays: HEK-293 cell lines stably transfected with a cyclicnucleotide-gated channel and either human CB-1 or CB-2 receptors (BDBiosciences, San Jose, Calif. USA) were seeded in poly-D-lysine coated96-well plates at a density of 70,000 cells per well. Plates wereincubated at 37° C. in 5% CO₂ overnight prior to assay. Plates were thenremoved from the incubator and the complete growth medium (DMEM, 10%FBS, 250 ∥g/ml G418 and 1 μg/ml puromycin) was replaced with 100 μL DMEMcontaining 0.25% BSA. Next, 100 μL membrane potential dye loading buffer(Molecular Devices, Sunnyvale, Calif. USA) was prepared according to themanufacturer. The plates were placed back into the incubator for 30minutes and then the baseline fluorescence was read on a BioTek Synergy2 multi-mode microplate reader (BioTek Instruments, Winooski, Vt. USA)with 540 nm excitation and 590 nm emission filters prior to drugaddition. Drugs were added in 50 μL DPBS containing 2.5% DMSO, 1.25 μM5′-(N-ethylcarboxamido) adenosine and 125 μM Ro 20-1724. Plates werethen incubated at room temperature for 25 minutes and fluorescencemeasured again at 540 nm excitation and 590 nm emission.

FIG. 1 depicts the functional activity of compound 6b at the CB-1receptor.

Cytoxocity assay: Cells were seeded on a 96 well polystyrene plate infull serum media at a density of 75,000 cells per milliliter, 100 μL perwell. Plates were incubated at 37° C. and 5% CO₂ for 24 hours to allowcell attachment. Drug solutions were prepared in DMSO at 100×concentration and mixed 1:100 in 1% FBS media to yield the desiredconcentration. Drug-media mixtures were vortexed immediately prior toadministration to cells. Full serum media was removed and replaced withdrug-media mixtures and incubated for 18 hours. 10 μL of Cell CountingKit 8 (CCK8, Dojindo# CK04-11) was added to each well to colormetricallyassess viability. After 2-4 hours of incubation with the CCK8 dye,absorbance was read at 450 nm by a BioTek Synergy 2 plate reader.

The cytotoxicity of selected compounds against the glioblastoma braincancer cell line LN-229 is depicted in Table 1.

TABLE 1 Compound EC₅₀ (μM) 6b 2.43 6c 1.83Inflammation Studies

Differentiation of Monocytes: To THP-1 human leukemia monocytes (ATCC#TIB-202) in suspension was added phorbol 12-myristate 13-acetate (PMAAldrich #P1585) and ionomycin (Aldrich #I0634), 10 and 500 ng/mlrespectively, to induce differentiation into macrophage-like cells.Cells were seeded at 30,000 cells/well and allowed to incubate at 37° C.in 5% CO₂/95% air for 3-10 days to complete transformation. Media wasrefreshed as needed until assay.

Cytokine Assay: A549 (ATCC #CCL-185), HUV-EC-C (ATCC #CRL-1730), ordifferentiated THP-1 cells were seeded on 96-well polystyrene plates ata density of 300,000 cells/ml (100 μL per well) and incubated at 37° C.in 5% CO₂/95% air for 24 hours to allow cell attachment. Drug solutionswere prepared in DMSO at 100× concentration and mixed 1:100 in 1% FBSmedia to yield the desired concentration.

Plates were then removed from the incubator and the complete growthmedia was replaced with 50 μL media containing 1% FBS andlipopolysaccharide or peptidoglycan at 1 μg/ml (for differentiatedTHP-1), or TNF-α (10 ng/ml) or IL-1β (1 ng/ml) in the case of A549 andHUVEC or without stimulus in the case of control wells. Cells werereturned to the incubator for 1 hour before drug treatments. Drug-mediasolutions were prepared at 2× desired final concentration in mediacontaining 1% FBS and the appropriate stimulus at the previouslymentioned concentration. Control media was also prepared which containedno drug. 50 μL of drug containing media or control was then added toappropriate wells and the plates returned to the incubator for 18 hours.Media supernatants were then removed from the wells and frozen at −80°C. until time of assay.

FIGS. 2-12 depict secretion profiles of various modulators by A549exposed to compound 6b at the EC1 and EC10 in the presence and absenceof TNF-α at 4 and 18 hour intervals. The graph legends are as follows:pyrC 1-4=6b at 1.1 μM for 4 hours; pyrC 2-4=6b at 1.6 μM for 4 hours;pyrC 1-18=6b at 1.1 μM for 18 hours; pyrC 2-18=6b at 1.6 μM for 18hours; TNF=TNF-α at 10 ng/ml.

The invention and the manner and process of making and using it are nowdescribed in such full, clear, concise and exact terms as to enable anyperson skilled in the art to which it pertains, to make and use thesame. It is to be understood that the foregoing describes preferredembodiments of the present invention and that modifications may be madetherein without departing from the spirit or scope of the presentinvention as set forth in the claims. To particularly point out anddistinctly claim the subject matter regarded as the invention, thefollowing claims conclude this specification.

What is claimed is:
 1. A compound of the formula

wherein: ----- represents an optional double bond wherein the ring thatoptionally contains it is selected from hexahydro, 6a,10a-dehydro,8,9-dehydro, and 9,10-dehydro; Y is selected from CH₂, CH(CH₃), CH(OH),C(CH₃)(OH), C(CH₃)₂, C(—U(CH₂),U—) and C(O); n is an integer ≧1; U isCH₂; Z is selected from H, substituted and unsubstituted alkyl, andcycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, and heterocycloalkylalkyl, whereineach alkyl portion is optionally substituted up to three tunes and thering portion of each is optionally substituted with one, two, three,four or five substituents; and R₁ is selected from H and substituted andunsubstituted alkyl.
 2. A compound according to claim 1 wherein Y isselected from carbonyl, dimethylmethylene and hydroxymethylene; and Z isselected from substituted or unsubstituted alkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted phenyl, andsubstituted or unsubstituted thiophenyl.
 3. A compound according toclaim 2 wherein Z is alkyl, cycloalkyl or phenyl.
 4. A compoundaccording to claim 3 wherein Z is hexyl, cyclohexyl or phenyl.
 5. Acompound according to claim 2 wherein Y is dimethylmethylene.
 6. Acompound according to claim 1 selected from (6aS, 9R,10aR)-6,6,9-trimethyl-3-(2-methyloctan-2-yl)-6a,7,8,9,10,10a-hexahydro-6H-isochromeno[4,3-c]pyridin-1-ol;(6aR, 9S,10aS)-6,6,9-trimethyl-3-(2-methyloctan-2-yl)-6a,7,8,9,10,10a-hexahydro-6H-isochromeno[4,3-c]pyridin-1-ol;((6aS, 9R,10aR)-3-(2-cyclohexylpropan-2-yl)-6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-isochromeno[4,3-c]pyridin-1-ol;(6aR, 9S,10aS)-3-(2-cyclohexylpropan-2-yl)-6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-isochromeno[4,3-c]pyridin-1-ol;(6aS, 9R,10aR)-6,6,9-trimethyl-3-(2-phenylpropan-2-yl)-6a,7,8,9,10,10a-hexahydro-6H-isochromeno[4,3-c]pyridin-1-ol;and (6aR, 9S,10aS)-6,6,9-trimethyl-3-(2-phenylpropan-2-yl)-6a,7,8,9,10,10a-hexahydro-6H-isochromeno[4,3-c]pyridin-1-ol.7. A pharmaceutical composition comprising a compound of claim
 1. 8. Apharmaceutical composition of claim 7 wherein the compound is selectedfrom the group consisting of (6aS, 9R,10aR)-6,6,9-Trimethyl-3-(2-methyloctan-2-yl)-6a,7,8,9,10,10a-hexahydro-6H-isochromeno[3,4-b]pyridin-1-ol;(6aR, 9S,10aS)-6,6,9-Trimethyl-3-(2-methyloctan-2-yl)-6a,7,8,9,10,10a-hexahydro-6H-isochromeno[3,4-b]pyridin-1-ol;((6aS, 9R,10aR)-3-(2-Cyclohexylpropan-2-yl)-6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-isochromeno[3,4-b]pyridin-1-ol;(6aR, 9S,10aS)-3-(2-Cyclohexylpropan-2-yl)-6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-isochromeno[3,4-b]pyridin-1-ol;(6aS, 9R,10aR)-6,6,9-Trimethyl-3-(2-phenylpropan-2-yl)-6a,7,8,9,10,10a-hexahydro-6H-isochromeno[3,4-b]pyridin-1-ol;and (6aR, 9S,10aS)-6,6,9-Trimethyl-3-(2-phenylpropan-2-yl)-6a,7,8,9,10,10a-hexahydro-6H-isochromeno[3,4-b]pyridin-1-ol.